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The Impact of the RBM4-Initiated Splicing Cascade on Modulating The www.nature.com/scientificreports OPEN The impact of the RBM4-initiated splicing cascade on modulating the carcinogenic signature of colorectal Received: 11 November 2016 Accepted: 06 February 2017 cancer cells Published: 09 March 2017 Jung-Chun Lin1, Yuan-Chii Lee2,*, Yu-Chih Liang1,*, Yang C. Fann3, Kory R. Johnson3 & Ying-Ju Lin4 A growing body of studies has demonstrated that dysregulated splicing profiles constitute pivotal mechanisms for carcinogenesis. In this study, we identified discriminative splicing profiles of colorectal cancer (CRC) cells compared to adjacent normal tissues using deep RNA-sequencing (RNA-seq). The RNA-seq results and cohort studies indicated a relatively high ratio of exon 4-excluded neuro- oncological ventral antigen 1 (Nova1−4) and intron 2-retained SRSF6 (SRSF6+intron 2) transcripts in CRC tissues and cell lines. Nova1 variants exhibited differential effects on eliminating SRSF6 expression in CRC cells by inducing SRSF6+intron 2 transcripts which were considered to be the putative target of alternative splicing-coupled nonsense-mediated decay mechanism. Moreover, the splicing profile of vascular endothelial growth factor (VEGF)165/VEGF165b transcripts was relevant to SRSF6 expression, which manipulates the progression of CRC calls. These results highlight the novel and hierarchical role of an alternative splicing cascade that is involved in the development of CRC. Alternative splicing constitutes a major mechanism in expanding the proteome diversity of eukaryotic cells1. Recent studies demonstrated that more than 90% of protein-coding genes generate more than one transcript through this meticulously controlled process2. In normal tissues, spatiotemporal expression profiles of alternative splicing events determine cell differentiation and specification3, whereas imbalanced or aberrant alternative splic- ing profiles are highly relevant to hereditary diseases and cancers4,5. The interplay between splicing factors, such as serine-arginine (SR) or the heteronuclear ribonucleoprotein (hnRNP) protein family, and cis-elements within cassette exons of alternative splicing events, constitutes the molecular mechanism of programming splicing pro- files6. Altered expressions or distributions of splicing factors were subsequently noted to be an efficient means for reprogramming splicing profiles of cancer cells7,8. Statistical studies indicated that while colorectal cancer (CRC) is the third most common and lethal cancer in the human race, the etiology and molecular mechanisms leading to CRC are largely unclear9. However, several alternative splicing events are widely observed and relevant to the development of CRC10. For instance, upreg- ulation of SRSF10 expression results in an increase in BCLAF1 exon 5a-included transcripts which facilitates the progression of CRC11. In addition, an increase in the expression of polypyrimidine tract-binding protein 1 (PTBP1) leads to a relatively high level of the PKM2 variant which mediates the Warburg effect in CRC cells12. Our previous work reported that overexpression of RNA-binding motif protein 4 (RBM4) constitutes a regulatory network in reprogramming splicing profiles of the FGFR2 and PKM genes, which modulate the progression and metabolic signature of CRC cells13. With the development of high-throughput approaches, including deep RNA sequencing (RNA-seq) and RNA crosslink immunoprecipitation-coupled proteomic analyses, alternative splicing networks can be investigated on a genome-wide level14,15. In this study, we performed RNA-seq analyses to thoroughly annotate transcriptomes in 1School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan. 2Graduate Institute of Biomedical Informatics, Taipei Medical University, Taipei, Taiwan. 3Information Technology and Bioinformatics Program, Division of Intramural Research, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA. 4School of Chinese Medicine, China Medical University, Taichung, Taiwan. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.-C.L. (email: [email protected]) SCIENTIFIC REPORTS | 7:44204 | DOI: 10.1038/srep44204 1 www.nature.com/scientificreports/ Figure 1. Transcriptome and gene ontology analyses of paired colorectal cancer (CRC) tissues. (a) Statistical summaries of transcriptome analytical results. (b) Transcriptome analysis-identified splicing events significantly changed in CRC tissues compared to adjacent normal tissues. (c) The top 11 enriched functions for differentially spliced genes in CRC tissues. CRC tissues and adjacent normal tissues. Among these alternative splicing events, RBM4, Nova1, SRSF6, and vas- cular endothelial growth factor 165 (VEGF165) comprised a splicing cascade in a consensus sequence-dependent manner which manipulated the migration and angiogenetic signature of CRC cells. These results suggested the potential value of splicing events as clinical applications for CRC treatment. Results Genome-wide analyses of CRC-associated alternative splicing events. Disclosing an imbalanced or aberrant splicing event may bring about a comprehensive understanding of the genetic causes of distinct malig- nancies. We therefore performed transcriptome analyses using deep RNA-seq with total RNAs extracted from cancerous and adjacent normal tissues of anonymous CRC patients. The statistical summary of the RNA-seq out- put showed close numbers of read lengths, amplified reads, and mapping rates within the independent analyses (Fig. 1a, n =​ 6). After filtering differentially spliced transcripts with a false detection rate (FDR)-adjusted p value of <​0.01, a q value of <​0.05, and a multiple of change of >​2, 153 genes from a total of 22,518 splicing events that significantly differed in cancerous tissues compared to adjacent normal counterparts were identified. Figure 1b presents 42 genes which exhibited differential splicing profiles between CRC cancerous tissues and adjacent nor- mal tissues. For instance, the relative level of the Nova1−4 transcript was significantly upregulated in cancerous tissues (Fig. 1b, 2.2237) compared to adjacent normal tissues (Fig. 1b, 0.3417). In contrast, relative expression of the non-coding SRSF6 transcripts in cancerous tissues (Fig. 1b, 79.3127) was more abundant than that of adjacent normal tissues (Fig. 1b, 13.0158). Gene ontology (GO) analyses were next conducted by examining putative CRC- related splicing events. Figure 1c shows the analytical summary (upper) and the bar chart (lower) presents the p values of the GO analyses. Results showed the enrichment of CRC-related splicing events involved in cytoskeletal, signaling receptor, and cell adhesion terms, which are highly related to the progression of cancer cells. Moreover, enriched terms corresponding to RNA metabolism and biosynthesis also implied a potential impact of RNA- binding proteins, including Nova1 and SRSF6 genes (Fig. 1b), on the carcinogenic signature of CRC cells. Differential expressions and splicing profiles of Nova1 and SRSF6 in CRC tissues. To validate the identified results regarding the splicing profiles of Nova1 and SRSF6 genes, RT-PCR analyses were conducted with RNAs prepared from cancerous tissues and adjacent normal tissues of anonymous CRC patients (Fig. 2a, n =​ 10). Figure 2a shows the predominant expression of exon 4-excluded Nova1 transcripts (Nova1−4, 60.1%, white bar) in cancerous tissues, whereas the reduced expression of Nova1−4 transcript (12.5%, white bar) was noted in the adjacent normal tissues of CRC patients. Furthermore, results of the RT-qPCR showed elevated total Nova1 tran- scripts in cancerous tissues compared to adjacent normal tissues (by about 4.4-fold; Fig. 2a, qRT-PCR). In addi- tion to Nova1, RNA-seq results also indicated differential splicing profiles of the SRSF6 gene between normal and cancerous tissues (Fig. 1b, lower table). The RT-PCR results showed that adjacent normal tissues exhibited pre- dominant expression of authentic SRSF6 transcripts (Fig. 2a, NM_006275; 87%, black bar) encoding functional SRSF6 proteins, whereas splicing profiles of the SRSF6 gene in cancerous tissues were shifted to non-coding SRSF6 SCIENTIFIC REPORTS | 7:44204 | DOI: 10.1038/srep44204 2 www.nature.com/scientificreports/ Figure 2. Differential splicing and expression profiles of Nova1 and SRSF6 in colorectal cancer (CRC) tissues and derived cell lines. (a) Total RNAs prepared from human CRC tissues (T) and adjacent non-tumorous tissues (NT) were subjected to RT-PCR and RT-qPCR analyses with specific primer sets complementary to the Nova1 and SRSF6 transcripts. The bar graphs present relative levels of the Nova1 and SRSF6 transcripts (left) or total Nova1 transcripts (right) in paired CRC tissues (n =​ 10). (b) Total lysates prepared from human CRC tissues (T) and adjacent non-tumorous tissues (NT) were analyzed using an immunoblot assay with the indicated antibodies. The bar graph presents relative levels of Nova1 and SRSF6 proteins in paired CRC tissues (n =​ 8). (c) Total RNAs and lysates prepared from CRC-derived cell lines were analyzed by RT-PCR assays with a specific primer set against the Nova1 and SRSF6 genes. Western blotting was performed with the indicated antibodies. The bar graph presents relative levels of the Nova1+4 and SRSF6 transcripts in three independent experiments. The gels shown in this figure were run under the same conditions and were
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